Methods of Collecting and Analyzing Dust Samples for Surveillance of Viral Diseases

This application is a national stage application filed under 35 U.S.C. § 371 of PCT/US2022/024483, filed Apr. 12, 2022, which claims the benefit of U.S. Provisional Patent Application Ser. No. 63/224,667 filed Jul. 22, 2021, the disclosure of which is expressly incorporated herein by reference in its entirety.

This invention was made with government support under grant/contract number R21 AI168817 awarded by the National Institutes of Health. The government has certain rights in the invention.

Applicant submits herewith a Sequence Listing in computer readable form and in compliance with 37 C.F.R. § 1.821-1.825. This sequence listing is in ASCII TXT format with filename “103361-119US1_2024_08_30_Sequence Listing,” a 2,073 bytes file size, and creation date of Aug. 28, 2024. The content of the Sequence Listing is hereby incorporated by reference.

COVID-19 has caused tens of millions of cases and millions of death since reaching pandemic designation in March 2020. The disease is spread by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Often, individuals are not symptomatic but may still spread the virus. Infected individuals spread the virus into the environment in respiratory droplets. Many droplets may land on the floor and other indoor surfaces.

We need new and improved methods to monitor for COVID-19 and other viral illnesses. We need techniques that could work at the building scale and complement individual testing and ongoing wastewater monitoring efforts. Wastewater samples can be difficult to collect and process, and not all individuals shed virus in feces. We need a more targeted approach to monitor for viruses in the indoor environment.

Described herein are methods for the detection of a virus (e.g., SARS-CoV-2) nucleic acid (e.g., viral RNA or viral DNA) in dust, which can be used for continued environmental surveillance of the viral disease (e.g., the novel coronavirus, SARS-CoV-2). Targeted monitoring of dust in high-concern buildings can complement broader population-level monitoring approaches. The examples described herein demonstrate that indoor dust can be used as a matrix for viral surveillance. Additionally, a method for detection of a viral nucleic acid (e.g., viral RNA or viral DNA) in a dust sample is disclosed herein.

Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. As used in the specification, and in the appended claims, the singular forms “a,” “an,” “the” include plural referents unless the context clearly dictates otherwise. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. The terms “optional” or “optionally” used herein mean that the subsequently described feature, event or circumstance may or may not occur, and that the description includes instances where said feature, event or circumstance occurs and instances where it does not. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, an aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. While implementations will be described for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monitoring in bulk floor dust and related samples, it will become evident to those skilled in the art that the implementations are not limited thereto, but are applicable for using indoor dust as a matrix for viral surveillance for other viruses of concern such as influenza A and B, respiratory syncytial virus (RSV), Rhinoviruses, Adenoviruses, and other emerging viral disease, such as Ebola/Marburg, Dengue, and Arenaviruses. Additional viruses may include but are not limited to Norwalk-like virus (norovirus), adenovirus, rhinovirus, other coronaviruses, parainfluenza, and others.

As used herein, the terms “about” or “approximately” when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer of this invention can be comprised of naturally occurring dNMP (i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide or non-natural nucleotide. The primer can also include ribonucleotides. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact length of the primers will depend on many factors, including temperature, application and source of primer. The term “annealing” or “priming” as used herein refers to the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, whereby said apposition enables the polymerase to polymerize nucleotides into a nucleic acid molecule which is complementary to the template nucleic acid or a portion thereof.

The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed 20 of deoxyribonucleotides.

The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.

The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers.

“Polymerase chain reaction”, or “PCR”, generally refers to a method for amplification of a desired nucleotide sequence in vitro. Generally, the PCR process consists of introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising a sample having the desired target sequence(s), where the primers are complementary to opposite strands of the double stranded target sequence. The reaction mixture is subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the desired target sequence flanked by the DNA primers.

The term “dust” refers to the combination of particulate matter and debris that accumulates naturally in buildings on the floor and on surfaces over time. Dust is also present in the air.

Environmental surveillance to assess pathogen presence within a community is proving to be a critical tool to protect public health, and it is especially relevant during the ongoing COVID-19 pandemic. Importantly, environmental surveillance tools also allow for the detection of asymptomatic disease carriers and for routine monitoring of a large number of people as has been shown for SARS-CoV-2 wastewater monitoring. However, additional monitoring techniques are needed to screen for outbreaks in high-risk settings such as congregate care facilities. The examples described herein demonstrate that SARS-CoV-2 can be detected in bulk floor dust collected from rooms housing infected individuals. This analysis suggests that dust is a useful and efficient matrix for routine surveillance of viral disease.

SARS-CoV-2 was measured using quantitative reverse transcription PCR (RT-qPCR), chip-based digital PCR (dPCR), and droplet digital PCR (ddPCR) in samples of bulk dust, passive surface samples, and surface swabs from rooms of individuals with COVID-19. In bulk dust, the SARS-CoV-2 viral concentration had a geometric mean value of 163 copies/mg of dust and ranged from nondetects to 23,049 copies/mg of dust (). SARS-CoV-2 RNA was detected in 89% of bulk dust, 55% of surface swabs, and 21% of passive surface sampler samples (average among all three detection methods used, Kruskal-Wallis P=0.02). The ddPCR method detected viral RNA in 97% of bulk dust samples compared to 93% for the chip-based dPCR and 76% for RT-qPCR (Kruskal-Wallis P=0.37) (). Across all sample types, the ddPCR method detected viral RNA in 60% of samples compared to 71% for the chip-based dPCR and 29% for RT-qPCR (Kruskal-Wallis P=0.06).

The COVID-19 isolation rooms were treated with a chlorine-based disinfectant prior to dust collection as part of the normal cleaning process, and the disinfectant is expected to largely inactivate the virus through reactions with the viral capsid (15). The bags were stored in the laboratory at room temperature after collection. Triplicate subsamples were extracted, and viral RNA was measured immediately upon collection and once per week for 4 weeks. Viral RNA did not measurably decay over 4 weeks in the vacuum bags (regression R=0.009, P=0.47) (). The coefficient of variance (CV) for number of copies/mg of dust ranged from 73.5% to 313.4% within each vacuum bag when averaged across the three methods of viral detection. This large variation in viral concentration is likely due to the heterogeneous mixture in the bags ().

The novel coronavirus and the ongoing COVID-19 pandemic have highlighted the need for sensitive and scalable viral surveillance within communities. In the long term, the threat of COVID-19 outbreaks will subside to a level where indefinite routine testing of asymptomatic individuals may be too cumbersome or expensive. However, there will continue to be a need to more broadly monitor vulnerable populations such as those in long-term-care facilities or high-risk patients in hospitals for SARS-CoV-2, influenza, respiratory syncytial virus (RSV), and other emerging viral diseases. Novel pathogens can be targeted with adaptable PCR-based assays. After detection, outbreaks can then be addressed with more targeted resources such as direct patient testing.

The results herein demonstrate that environmental dust collection can provide a convenient and useful matrix for ongoing viral monitoring. The process can provide monitoring for many high-risk individuals, and dust samples are already being collected through normal cleaning practices such as vacuuming. Dust had a higher positivity rate than surface swab samples, and the positivity rate of the surface swabs in this study was similar to or greater than the rates in similar studies (16, 17). Our observations indicate that SARS-CoV-2 RNA in dust can persist at least 4 weeks after dust collection and that the measured concentration can vary in different dust subsamples within a vacuum bag. Therefore, multiple samples should be taken from a bag to more rigorously quantify the viral genetic signal, or homogenization methods should be developed that comply with biosafety standards. Additionally, RNA and dust persistence in the environment should be considered when determining if the outbreak occurred recently or in the past. Differences between PCR-based measurement methods may inform method choice. For instance, RT-qPCR requires calibration standards for quantification and the digital methods do not, and for the assays used, ddPCR is a one-step reaction and the chip-based dPCR requires a two-step reaction. Each instrument also has a different detection limit and resulted in marginally different positivity rates. Previously, measurements of indoor environmental microbes have been used to detect infectious microbes such asand(18-20). However, nucleotide-based tests do not measure infectivity, meaning the detection of genetic material from these microbes may indicate that people in the area are infected but would not necessarily indicate the risk of infection due to contact with indoor surfaces or via resuspension of floor dust.

Indoor dust can also be used to complement other environmental surveillance methods, e.g., wastewater monitoring. Wastewater detection may be more beneficial at larger population scales covering thousands of individuals in a community, and one infected individual can be detected among 100 to 2,000,000 individuals (21). Indoor dust can be useful in areas with smaller numbers of high-risk individuals where more specific outbreak identification is critical. Additionally, not all individuals secrete virus in stool (22). Indoor dust sampling can also be less expensive and be easier to implement, with simplified sample collection and no preconcentration steps of samples required. Other dust collection methods are available beyond those described in this study. Future research should evaluate differences between collection strategies.

Indoor dust provides an important matrix for environmental surveillance of viral disease outbreaks. Infected humans shed virus into their surrounding environment, which becomes integrated into the dust. In many cases, dust is already being collected during routine cleaning and can easily be submitted for analysis. Overall, dust can be a useful and efficient matrix to provide identification of viral disease in high-risk settings, such as congregate care facilities. Future research can validate these results on a broader scale and in different building types to better inform use of this technique to mitigate viral transmission.

An example method for environmental surveillance is described below. The method includes collecting a dust sample from an area inside an enclosed structure. This disclosure contemplates that the enclosed structure is a building such as a house, hotel/motel, school, office, medical facility, etc. As described herein, the dust sample is a bulk dust sample in some implementations. In other implementations, the dust sample is a surface swab sample or a passive surface sample. Optionally, the method includes sieving the dust sample (e.g., to 250 μm or 300 μm in diameter or any other size). The method also includes extracting viral RNA from the dust sample. Techniques for extracting viral RNA from the dust sample are described below. It should be understood that many viruses will likely not be viable when measured (especially for enveloped viruses). See Nicholas Nastasi et al., “Viability of MS2 and Phi6 Bacteriophages on Carpet and Dust,” doi.org/10.1101/2021.05.17.444479. This offers a distinct advantage because 1) the samples do not need to be stored cold and can be used at least out to 4 weeks after collection and 2) for enveloped viruses especially this reduces the biosafety hazard of handling the dust. Additionally, the method includes quantifying an amount of the extracted viral RNA contained in the dust sample. For example, this can be accomplished by performing a PCR assay on the extracted viral RNA. As described herein, the PCR assay is a RT-qPCR assay in some implementations. In other implementations, the PCR assay is a digital PCR assay (e.g., dPCR or ddPCR assay). Example PCR assays are described below. It should be understood that PCR-based detection is provided only as an example. This disclosure contemplates using other techniques to quantify the amount of the extracted viral RNA contained in the dust sample. This may include using a lateral flow test or loop-mediated amplification (LAMP) technology, for example. Lateral flow assay is also described in the art, including U.S. Pat. No. 8,399,261; U.S. Publication No. 20150293086A1; each of which is herein incorporated by reference.

The method further includes determining whether a viral disease (e.g., SARS-CoV-2) is present inside the enclosed structure based on the amount of the extracted viral RNA contained in the dust sample. This disclosure contemplates that the determination can be performed by a computing device (e.g., computing device shown in). For example, the amount of the extracted viral RNA contained in the dust sample can be received at a computing device and then compared to a threshold value. If the amount of the extracted viral RNA contained in the dust sample exceeds the threshold value, then the viral disease is present inside the enclosed structure. Otherwise, the viral disease is not present inside the enclosed structure. In some implementations, there are a plurality of threshold values, each representing a different level of risk. For example, samples<1 copies/mg dust are “green,” samples 1-<10 copies/mg dust are “yellow,” and samples>=10 copies/mg dust are “red”. It should be understood that <1, 1-<10, >=10 are threshold values.

In one example implementation where the viral disease is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) including delta and omicron variants thereof, the threshold value can be between about 50 copies/mg dust and about 1000 copies/mg dust. For example, the threshold value may be about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 copies/mg dust. Optionally, the threshold value can be between about 200 copies/mg dust and about 400 copies/mg dust. For example, the threshold value may be about 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 copies/mg dust. Optionally, the threshold value can be between about 300 copies/mg dust. For example, the threshold value may be about 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, or 325 copies/mg dust. In some embodiments, SARS-CoV-2 is an alpha variant (e.g., B.1.1.7 or Q lineages), a beta variant (e.g., B.1.351 or descendent lineages), a gamma variant (e.g., P.1 and descendent lineages), an epsilon variant (e.g., B.1.427 or B.1.429), an eta variant (e.g., B.1.525), an iota variant (e.g., B.1.526), a kappa (e.g., B.1.617.1), 1.617.3, a mu variant (e.g., B.1.621, B.1.621.1), a Zeta variant (e.g., P.2), a Delta variant (e.g., B.1.617.2 or AY lineages), or an omicron variant (e.g., B.1.1.529 or BA lineages). In some examples, disclosed herein is a method of detection and/or monitoring any variants of SARS-CoV-2.

In some embodiments, the method disclosed herein further comprises disinfecting the tested area and/or the dust sample if the amount of the detected viral nucleic acid in the dust sample is above the threshold value. In some embodiments, the threshold value is between about 50 copies per milligram (mg) dust and about 1000 copies per mg dust. In some embodiments, the threshold value is about 300 copies per mg dust.

It should be understood that the threshold value will vary with the type of viral disease. In some implementations, the threshold value may be the detection threshold (i.e., detected/not detected), for example, for a deadly disease such as the Ebola virus. While in other implementations, the threshold value is greater than the detection threshold and also specific to the viral disease. Thus, the threshold value for viral diseases other than SARS-CoV-2 may be different than the example threshold values provided above.

Optionally, an action item from a risk management plan can be recommended based on the comparison. This disclosure contemplates that the recommendation can be provided by a computing device (e.g., computing device shown in). For example, the action item is to test individuals for the viral disease. Alternatively or additionally, the action item is to take a personal protective measure and/or use personal protective equipment. Alternatively or additionally, the action item is to sequence the extracted viral RNA to detect variants. It should be understood that the individual testing and sequencing are provided only as example action items. This disclosure contemplates that the risk management plan can include more, less, and/or different action items than the examples. For example, action items may include, but are not limited to, mandated masking, social distancing, building lockdown, enhanced cleaning, and improved ventilation.

Optionally, a report including the amount of the extracted viral RNA contained in the dust sample can be generated. Heatmaps such as those shown inand RNA concentration and cumulative norm probability plots shown inare example reports. Alternatively or additionally, reports similar to those shown by(heatmap for environmental surveillance of structures over time) and(percentage of variants in dust samples) can be provided. This disclosure contemplates that the report can be generated by a computing device (e.g., computing device shown in). It should be understood thatare provided only as example reports. This disclosure contemplates that the reports can be other than those shown in the figures. For example, a report may include color-coded buildings on a map, where different colors indicate respective viral concentrations within the buildings. Optionally, an action item from a risk management plan can be recommended based on the report. For example, this disclosure contemplates that reports similar to those shown byand/orcan be used to understand the spread of the viral disease by location and variant over time and such information may be used to inform actions.

Optionally, the amount of the extracted viral RNA contained in the dust sample can be correlated to an approximate number of infected individuals using the enclosed structure. This disclosure contemplates that the correlation can be performed by a computing device (e.g., computing device shown in).

Optionally, the method further includes sequencing viral RNA that has been extracted from the dust sample to detect variants. Techniques for sequencing viral RNA are described herein.

Disclosed herein is a method for detection of a viral nucleic acid in a dust sample, comprising

In some embodiments, the viral nucleic acid is a viral RNA or viral DNA. Methods for nucleic acid extraction are known in the art. See, e.g., U.S. Pat. Nos. 9,738,931 and 9,580,751, incorporated by reference herein in their entireties. In some examples, step a) comprises phenol-based lysis.

Step a) of the method disclosed herein comprises using an RNase inhibitor, wherein the RNase inhibitor can be any composition known in the art that can inactivate, denature, and/or inhibit a ribonuclease. Beta-mercaptoethanol (BME) is a reducing agent that can irreversibly denature RNases. In some examples, step a) of the method disclosed herein comprises using a concentration of beta-mercaptoethanol at least about 1.5 times greater (for example, at least about 1.5 times greater, at least about 2 times greater, at least about 3 times greater, at least about 4 times greater, at least about 5 times greater, at least about 6 times greater, at least about 7 times greater, at least about 8 times greater, at least about 9 times greater, at least 10 times greater, at least about 20 times greater, at least about 40 times greater, at least about 50 times greater, or at least 100 times greater) than a recommended concentration. In some embodiments, step a) comprises using a concentration of beta-mercaptoethanol about 10 times greater than a recommended concentration. Dust contains high concentrations of RNases that must be denatured with beta-mercaptoethanol or other protein denaturing compounds prior to extraction. The recommended concentration of beta-mercaptoethanol can be, for example, about 1%, 2%, 5%, 10%, or 20% of a lysis buffer. In some embodiments, the concentration of beta-mercaptoethanol used in the method disclosed herein is about 10%, 20%, or 50% of a lysis buffer (v/v). In some embodiments, the concentration of beta-mercaptoethanol used in the method disclosed herein is about 8% to about 12% or about 9% to about 11% of a lysis buffer (v/v). In some embodiments, the concentration of beta-mercaptoethanol used in the method disclosed herein is about 10% of a lysis buffer (v/v). In some embodiments, an alternative compound that denatures proteins and specifically RNases can be used.

In some examples, step b) of the method disclosed herein comprises

In some examples, the level of inhibition is increased if the amplification of the nucleic acid template is delayed by at least about 1 cycle (for example, at least about 1.2 cycles, at least about 1.4 cycles, at least about 1.6 cycles, at least about 1.8 cycles, at least about 2 cycles, at least about 4 cycles, at least about 10 cycles, at least about 50 cycles, or at least about 100 cycles). The term “reference control” refers to a level detected in a sample without the addition of the viral nucleic acid (e.g., viral RNA or viral DNA) extracted from the dust sample.

The technique of PCR is described in numerous publications, including, PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications, by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584, each of which is herein incorporated by reference. In some examples, the PCR assay is a quantitative reverse transcription PCR (RT-qPCR) assay or a digital PCR assay (for example, a chip-based digital PCR (dPCR) or droplet digital PCR (ddPCR) assay).

In some examples, the method disclosed herein further comprises sequencing the viral nucleic acid (e.g., viral RNA or viral DNA) extracted from the dust sample. The sequencing techniques are known in the art, including, for example, Maxam-Gilbert sequencing, chain-termination methods, shotgun sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, short-read sequencing methods, massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, illumina (Solexa) sequencing, combinatorial probe anchor synthesis (cPAS), SOLID sequencing, lon Torrent semiconductor sequencing, DNA nanoball sequencing, or heliscope single molecule sequencing. In some examples, the viral nucleic acid (e.g., viral RNA or viral DNA) is reverse transcribed and amplified by PCR before the step of sequencing.

In some examples, the viral RNA or DNA with a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. In some embodiments, the viral RNA is a SARS-CoV-2 RNA.

In some examples, the method disclosed herein comprises the steps in the process:

The commercial kit used for the extraction step can be the Qiagen RNeasy Powermicrobiome extraction kit or other extraction methods can be used. The following modifications are made:

For sequencing, the extraction described above is critical to have the highest-quality template. Samples can be amplified in triplicate and the products pooled prior to sequencing to provide higher rates of success. Trying different dilutions and adding BSA can also be helpful.

Sars-CoV-2 Sequencing from Dust

It should be appreciated that the logical operations described herein with respect to the various figures may be implemented (1) as a sequence of computer implemented acts or program modules (i.e., software) running on a computing device (e.g., the computing device described in), (2) as interconnected machine logic circuits or circuit modules (i.e., hardware) within the computing device and/or (3) a combination of software and hardware of the computing device. Thus, the logical operations discussed herein are not limited to any specific combination of hardware and software. The implementation is a matter of choice dependent on the performance and other requirements of the computing device. Accordingly, the logical operations described herein are referred to variously as operations, structural devices, acts, or modules. These operations, structural devices, acts and modules may be implemented in software, in firmware, in special purpose digital logic, and any combination thereof. It should also be appreciated that more or fewer operations may be performed than shown in the figures and described herein. These operations may also be performed in a different order than those described herein.

Referring to, an example computing deviceupon which the methods described herein may be implemented is illustrated. It should be understood that the example computing deviceis only one example of a suitable computing environment upon which the methods described herein may be implemented. Optionally, the computing devicecan be a well-known computing system including, but not limited to, personal computers, servers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, network personal computers (PCs), minicomputers, mainframe computers, embedded systems, and/or distributed computing environments including a plurality of any of the above systems or devices. Distributed computing environments enable remote computing devices, which are connected to a communication network or other data transmission medium, to perform various tasks. In the distributed computing environment, the program modules, applications, and other data may be stored on local and/or remote computer storage media.

In its most basic configuration, computing devicetypically includes at least one processing unitand system memory. Depending on the exact configuration and type of computing device, system memorymay be volatile (such as random access memory (RAM)), non-volatile (such as read-only memory (ROM), flash memory, etc.), or some combination of the two. This most basic configuration is illustrated inby dashed line. The processing unitmay be a standard programmable processor that performs arithmetic and logic operations necessary for operation of the computing device. The computing devicemay also include a bus or other communication mechanism for communicating information among various components of the computing device.

Computing devicemay have additional features/functionality. For example, computing devicemay include additional storage such as removable storageand non-removable storageincluding, but not limited to, magnetic or optical disks or tapes. Computing devicemay also contain network connection(s)that allow the device to communicate with other devices. Computing devicemay also have input device(s)such as a keyboard, mouse, touch screen, etc. Output device(s)such as a display, speakers, printer, etc. may also be included. The additional devices may be connected to the bus in order to facilitate communication of data among the components of the computing device. All these devices are well known in the art and need not be discussed at length here.